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Specific human and Candida cellular interactions lead to controlled or persistent infection outcomes during granuloma-like formation

Infection and Immunity (2017) 85(1)
DOI: 10.1128/IAI.00807-16
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Abstract

A delayed type of multicellular process could be crucial during chronic candidiasis in determining the course of infection. This reaction, consisting of organized immune cells surrounding the pathogen, initiates an inflammatory response to avoid fungal dissemination. The goal of the present study was to examine, at an in vitro cellular scale, Candida and human immune cell interaction dynamics during a long-term period. By challenging human peripheral blood immune cells from 10 healthy donors with 32 Candida albicans and non-albicans (C. glabrata, C. tropicalis, C. parapsilosis, C. dubliniensis, C. lusitaniae, C. krusei, and C. kefyr) clinical isolates, we showed that Candida spp. induced the formation of granuloma-like structures within 6 days after challenge, but their sizes and the respective fungal burdens differed according to the Candida species. These two parameters are positively correlated. Phenotypic characteristics, such as hypha formation and higher axenic growth rate, seem to contribute to yeast persistence within granuloma-like structures. We showed an interindividual variability of the human response against Candida spp. Higher proportions of neutrophils and elevated CD4+/CD8+ T cell ratios during the first days after challenge were correlated with early production of gamma interferon (IFN-γ) and associated with controlled infection. In contrast, the persistence of Candida could result from upregulation of proinflammatory cytokines such as interleukin-6 (IL-6), IFN-γ, and tumor necrosis factor alpha (TNF-α) and a poor antiinflammatory negative feedback (IL-10). Importantly, regulatory subsets of NK cells and CD4lo CD8hi doubly positive (DP) lymphocytes at late stage infiltrate granulomalike structures and could correlate with the IL-10 and TNF-α production. These data offer a base frame to explain cellular events that guide infection control or fungal persistence.

Figures

  • FIG 1 In vitro immune infiltrates induced by Candida spp. after infection of human immune cells from immunocompetent subjects. (A) Representative immune infiltrates observed under light microscopy 6 days after challenge of human mononuclear and polymorphonuclear peripheral blood cells with living yeasts from annotated Candida species (MOI, phagocyte-to-yeast ratio of 2,000:1). Bars represent 50 m. No formation of immune infiltrates was observed in uninfected conditions for up to 6 days postinfection (p.i.). (B) Number of immune infiltrates per cubic centimeter on days 4 and 6 postinfection by the eight Candida species. Each dot represents the mean of the number of structures per cubic centimeter for one clinical isolate and for all studied subjects. Lines indicate SEM (*, P between 0.045 and 0.0360; **, P 0.0017; ***, P between 0.0004 and 0.0002; ****, P 0.0001; 0.05). C.alb, C. albicans; C.dub, C. dubliniensis; C.trop, C. tropicalis; C.lus, C. lusitaniae; C.gla, C. glabrata; C. par, C. parapsilosis; C.kru, C. krusei; C.kef, C. kefyr. (C) Box plots depict median, minimum, and maximum immune infiltrate numbers for each donor subject (S) on days 4 (white boxes) and 6 (gray boxes) postinfection (n 80).
  • FIG 2 Dynamics of immune infiltrate size according to Candida species. (A) Human mononuclear and polymorphonuclear peripheral blood cells from donors were infected with Candida spp. for up to 6 days. The size of immune infiltrates was measured by light microscopy on days 4 and 6 postinfection. The data are presented as the mean SEM of structure sizes from 10 subjects for 32 clinical isolates of Candida species 6 days postinfection. (B) Coculture analysis by time-lapse video imaging. Human peripheral blood mononuclear and polymorphonuclear cells were infected with C. albicans cells at an MOI of 2,000:1. Cell aggregation was followed for 72 h, and a capture was done every 10 min. Bars represent 50 m. (C) Pairwise correlation of immune infiltrates size and fungal burden. Shown is a graphical representation of the fungal burdenmeans (x axis) and immune infiltrate size (y axis) 6 days postinfection. The line indicates the slope.
  • FIG 3 Dynamics of Candida proliferation within immune infiltrates. (A) The fungal burden was determined at 0, 2, 4, and 6 days postinfection. Data are the means SEM of the fungal burden of 32 clinical isolates of Candida from immune infiltrates of 10 subjects. The black line represents the mean SEM of the fungal burden of 32 clinical isolates of Candida and of 10 subjects. The gray dashed line represents the mean SEM of the fungal burden of C. albicans immune infiltrates, while the gray dotted line shows the mean SEM of the fungal burden of C. krusei immune infiltrates. (B) Generation time (G) and growth analysis of Candida species isolates. Shown are means SEM for generation time for Candida species. (C) Significant positive correlation between mean of growth test after 6 days and mean of fungal burden on day 6 after infection.
  • TABLE 1 Dynamics of fungal burden within granuloma-like structures for Candida species
  • FIG 4 Histological examination of immune infiltrates induced by Candida spp. (A) Light microscopy observation of immune infiltrates and cell migration gradients (CM) 6 days postinfection. Cells were washed twice in PBS, and remaining compact inflammatory aggregates (IA) were analyzed. Scale bar: 50 m. (B) May-Grünwald-Giemsa staining of immune aggregates 6 days after infection. Y, yeasts; PH, pseudohypha; H, hypha; Mf, activated macrophages; EC, large activated macrophages; MC, multinucleated cells with two nuclei; FM, foamy macrophages; MGC, multinucleated giant cells; N, neutrophils; Ly, lymphoid cells. Bars represent 20 m. (C) Time-lapse and confocal microscopy examination of granuloma-like structures. Human immune cells were incubated with GFP-tagged C. albicans under the same coculture conditions as described in the text. Nuclear DNA was stained with Hoechst. Fluorescence-stained sections were examined under a Nikon A1 RSI microscope at a magnification of 20 and constant Z-steps, and 3D images were processed with NIS elements version 3.21 and Volocity 3D image analysis software version 6.01.
  • FIG 5 Interindividual variability of the response against Candida spp. The variability of the subjects’ response against Candida infection was studied after infection of human peripheral blood mononuclear and polymorphonuclear cells with living yeasts from different Candida species. (A) Fungal burden 6 days postinfection. Results are expressed as the mean SEM of the fungal burden of 4 Candida clinical isolates from each species for each subject (S1 to S10). Dotted lines indicate the arbitrary cutoff of 100 CFU/ml. The mean SEM for each subject and each Candida species were compared to the mean SEM of species that controlled infection (mean of C. krusei and C. kefyr). *, P 0.05; **, P 0.001. (B) Number of subjects showing a persistent-infection (white bars) or controlled-infection (green bars) status.
  • FIG 6 Characterization of CD66 and CD14 cell proportions within Candida granuloma-like structures over time. Peripheral blood mononuclear and polymorphonuclear cells from 10 healthy subjects were infected with 32 Candida clinical isolates for up to 6 days. The granuloma-like structures were collected from coculture plates at different time points and stained with a cocktail of fluorescenceconjugated antibodies specific to CD66 neutrophils and CD14 monocytes. (A) Representative flow cytometry analysis showing a side-scatter (SSC) versus forward-scatter (FSC) plot of granulocyte (I) and monocyte (II) selection. CD66 cells were gated from region I, and CD14 monocytes were gated from region II. (B) The proportions of CD66 cells within granuloma-like structures are expressed as percentages of the total living cell compartment. Box plots depict median, minimum, and maximum percentages of CD66 cells in persistent-infection and controlled-infection subjects. (C) Scatter plot of CD66 proportions according to persistent-infection and controlled-infection status. (D) The proportions of CD14 cells within granuloma-like structures are expressed as a percentage of the total living cell compartment. Box plots depict median, minimum, and maximum percentages of CD14 cells according to persistent-infection and controlled-infection status. (E) Scatter plot of CD14 cells proportions. *, P 0.05; **, P 0.001; ***, P 0.0001; ****, P 0.00001 (by one-way ANOVA with Tukey’s multiple-comparison test) (n 320). ns, not significant.
  • FIG 7 Characterization of CD56 NK cells within Candida granuloma-like structures over time. Peripheral blood mononuclear and polymorphonuclear cells from 10 subjects were infected with 32 Candida clinical isolates for up to 6 days. The granuloma-like structures were collected from coculture plates at different time points and stained with a cocktail of fluorescence-conjugated antibodies specific to CD3 and CD56 lymphocytes. (A) Representative flow cytometry analysis showing SSC versus FSC plot of lymphocytes in section III. The cells were analyzed over time after gating on CD3 lymphocytes. Natural killer cells were gated as CD56 from the CD3 population. (B) The proportions of CD56 cells within granuloma-like structures are expressed as percentage of the total CD3 compartment. Box plots depict median, minimum, and maximum percentages of CD56 cells in persistent-infection and controlled-infection granuloma-like structures. (C) Scatter plot of CD56 NK cells proportions according to persistent- and controlled-infection status. *, P 0.05; **, P 0.001; ***, P 0.0001; ****, P 0.00001 (by one-way ANOVA with Tukey’s multiple-comparison test) (n 320).

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Misme-Aucouturier, B., Albassier, M., Alvarez-Rueda, N., & Pape, P. L. (2017). Specific human and Candida cellular interactions lead to controlled or persistent infection outcomes during granuloma-like formation. Infection and Immunity, 85(1). https://doi.org/10.1128/IAI.00807-16

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